A Review on the Sorghum for biofuel and microRNAs

Energy is lifeline in development and progress of a country, with pivotal use in industrial and agricultural sectors. While fossil fuels are primary source of energy, its rapid depletion is major concerns of the world today. Developed countries are finding alternates to strengthen its economic backbone by resolving limited supply of energy issues and meeting their enormous demands. Plants can offer better alternative as biofuels to address the energy crisis and meet projected energy demand. Recently, sorghum based research remained center of attention due to its exceptional properties to grow under dry, and hot environmental conditions with limited water requirement. All parts of sorghum have economic values with usage in syrup, sugar, fuel, alcohol, bedding and paper production. Whereas, Sorghum stalks are enriched source of carbohydrates with 16-18% of fermentable sugar that makes it potential candidate for bioethanol production. Bioethanol is considered environmental friendly as it reduces greenhouse gases and replaces MTBE (Methyl tert-butyl ether) pollutants in air. Yet, the benefits of sorghum as biofuels comes with a challenge of rapid degradation of sugar, therefore immediate harvest after maturity can ensure high sugar content. This review covers scope and recent research on sorghum in Pakistan and indicates its usage as an ideal feedstock to meet present energy crisis. Currently, developed countries are exploiting sorghum in bioethanol production due to its high tolerance to drought and salt, and improved sugar content in lieu of using sugarcane and maize. Moreover, studies involving high-tech research and role of microRNA in high yield and improved sugar content in biofuel production of sorghum are also addressed in this review.


Introduction Sorghum based research in Pakistan
Pakistan is an agriculture based country with variety of important cereal crops, including sorghum.Sorghum ranks fifth among important cereal crops of the world, and is top summer grass of Pakistan and commonly called as 'Jawar.'It is widely used as staple food for humans besides its usage as fodder for animals.Sorghum is warm-weather crop and adapted to wide range of soil and climate conditions [1].Its grain comprises of 70% carbohydrates, 10-12% protein and 3% fats, while fodder is highly enriched with prussic acid and oxalic acid, therefore grains can be introduced in feeding programs for poultry and cattle [2].Around 2.35 million hectares of land, contributing in 12% of the cultivated land is cropped area for fodder production in Pakistan (Agricultural Statistics of Pakistan, 2005).Though production of good quality forage requires well organized livestock industry, food crop land cannot be compensated for the fodder cultivation.Therefore, legumes are intercultured with cereal and forage crops to increase fodder supply.Although cereal crops give high yield, their protein content is lower than legumes [3].Yield of sorghum is higher even from small farm area as compared to other plants however, the forecasted values of sorghum production revealed decrease in cultivation trend in near future.This decrease in cultivation trend is due to improper use of sorghum for industrial purpose and use of low yielding varieties [4].Nevertheless, the research on sorghum has been increased in recent years while advancing from physio-morphological characteristics.
Previously, germplasm characterization was performed using physiological, morphological or isozyme markers.Recently, DNA finger printing has removed the barriers in germplasm characterization by providing enough genetic information that was unavailable at phenotypic level.Tabbasum and her coworkers [5] distinguished two sorghum hybrids while identifying 78.98% genetic resemblance among them using random amplified polymorphic DNA (RAPD) [5].Advancement in molecular techniques has helped identifying genetic diversity among crops and thus use of agronomic, physiological and morphological traits have been improvised to the level of biochemical and molecular markers.A study conducted by Mehmood et al. [6] approached phylogenetic relationship and genetic diversity of 10 sorghum varieties through RAPD analysis, and found 78.94% polymorphism.Through their work sorghum varieties, YSS-9 and 84G01 showed distant relationship, while RARI-S3 and RARI-S-4 were found to be closely related [6].Sorghum has diverse genome, therefore, genetic fingerprinting provides an efficient marker system.In 2010, another genetic diversity study on 29 sorghum varieties was conducted among exotic, and approved local lines using RAPD, revealing 95% polymorphism among the varieties.They found that the genotypes of exotic lines K-A-113 and Indian III exhibited maximum similarity, whereas F-606 and F-601 showed distinctive features [7].Till date genotypes of many crops are evaluated using physiological markers, as recent study [8] evaluated drought tolerance in sorghum (80 accession) using physiological parameters revealing significant differences in all accessions under stress.Osmotic pressure was considered potential trait for drought tolerance, while five accessions (80265, 80114, SS-95-4, SS-97-7 and 80377) were found widely tolerant to water stress [8].Furthermore, a study conducted on role of exogenous salicylic acid in salinity tolerance on post germination seedlings (PARI-S-4 and YSS-9) revealed that low level of salicylic acid reverse the impact of salinity in seedlings but at high salt concentration (50 mg L-1): beyond threshold causing high level of Na+/ K+ ratio, salicylic acid was found ineffective in reducing salinity [9].Another study on determining effect of exogenous proline in reducing impact of salinity in sorghum cultivars revealed that low concentration (50 mM) of proline boost physiological characteristics of sorghum, whereas as high concentration (100 mM) together with high salt concentration alleviated the adverse effect caused by salt stress [10].Akram et al. [11] determined genetic variability for drought tolerance in 20 genotypes of sorghum cultivars using 10 RAPD primers.Maximum similarity of 95.5% was noted between YSS-17 and PARC-SS-1 genotypes, while minimum similarity of 51.5% was observed for DS-97-1 genotype.Furthermore, maximum similarity was observed between YSS-10 (C) and YSS-18, YSS-98 and SV-10, CSV-15 and Rasili, VI-1 and RS-29 [11].In another study, 17 of sorghum landraces were evaluated for grain yield and drought tolerance using physio-morphological markers at seedling and post flowering stages, revealing high genetic difference in water stress tolerance for higher grain yield [12]. Hussain et al.
[13] evaluated eight genotypes of sorghum of Potohar region for various grain and associated traits, showing significant difference in grain yield, plant height, stalk yield, days to 50% flowering and maturity.Among tested varieties of sorghum, SPV-462 was stalk producing variety with higher grain yield, whereas Johar and CSP-15 SPV were high grain producing varieties, while SPV and YSS were high stalk producing varieties.Among the sorghum varieties tested, PARC-SS-2 took minimum days to 50% flowering, whereas, CSV-15 and YSS-9 took longest time to 50% flowering [13].Sher et al. [14] investigated the effect of harvest time associated with P and S fertilization on yield and quality of forage sorghum (Sorghum bicolor (L.) Moench).They concluded that harvesting at maturity stage along with P and S fertilization increases forage yield and other quality related traits as: 30% decrease in leaf hydrocyanic acid content, and 50% increase in stalk soluble-solid content widely used as an indicator of forage juiciness and palatability [14].Furthermore, a study [15] on effect of potash fertilizer dose on sorghum hybrid (Pioneer MR-Buster) and maize hybrid (Pioneer 3062) showed high grain yield with increase in potash level and number of split applications.Potash fertilizer caused significant differences in all parameters except for plant height, thus, highest grain yield was recorded in maize (8014 kgha-1) with three-split dose of 120 kgha-1, as compared to control with minimum grain yield.Therefore, recommended application of 120 kgha-1 of potash fertilizer with 3 splits can help improve grain yield for both crops [15].In Pakistan, salinity is among major challenges that adversely affect the yield and growth of crops to various extents.Therefore, cultivation of salinity tolerant lines can bring solution to obtain economical yield.Kausar et al.
[16] screened physiological parameters of sorghum lines to identify salinity tolerance.Among all sorghum lines, Sandalbar and JS-2002 were considered tolerant, while JS-263 and Hegari had medium tolerance.Sorghum line Noor was conceived as medium sensitive, whereas, PSV-4 and FJ-115 were found sensitive to salinity [16].Another study for effects of heavy metal (NiCl) on morphological characteristics of sorghum demonstrated that increasing concentration up to 90 ppm (of NiCl) can adversely reduce dry weight of sorghum while 30ppm was considered minimum threshold level [17].Seed priming technique widely used to stimulate the seed emergence and seedling growth has been studied by Shehzad et al.
[18] to evaluate effect of these different techniques such as: un-soaked seed (control), Halopriming with KNO3 and CaCl2 (1% solution), Hydro-priming (soaked with distill water) etc.On germination and seedling growth of three sorghum varieties (JS-2002, Hegari, and JS-263).All priming techniques accelerated germination rate by 50%, while Halopriming with KNO3 and CaCl2 helped improving seedling growth and sorghum emergence [18].Genetic potential of 20 sorghum genotypes was evaluated for drought tolerant traits (heritable and measurable) after artificially creating water stress via PEG treatment and five (80353, 80365, 80199, 80204 and 80319) were found superior to be used in drought tolerant breeding programs.Therefore, crops at early growth stages can be screened using different heritable morphological parameters for drought stress [19].Seven advanced lines of sorghum, i.e., Noor, Hegari, F-9917, F-207, F-214, JS-2002 and PC-1 were evaluated for nutritive profile as well as dry matter and forage material, and Hegari genotype was found superior in terms of forage material as well as nutritive dry matter due to high leaf area [20].F-9917 and F-214 displayed poor performance in terms of ash content, crude protein and fiber needed to be served for forage purpose.These finding on new genotypes revaled that genetic variation among sorghum genotypes can contribute in potential traits for forage purpose and therefore, can be exploited [20].Although hydrogen cyanide (HCN) is associated with it, sorghum is considered main forage crop for livestock.Zahid et al.
[21] determined the effects of HCN content at different growth stages (of plant, i.e., 3rd leaf (GS1), pre-booting (GS2) and 50% heading stage (GS3) and post cutting) for local sorghum cultivars: including JS-2002, and Chakwal sorghum.Sorghum JS-2002 was considered safe for forage purpose due to high crude protein percentage, lowest HCN content and, higher crude fiber at prebooting stage.Moreover, though the content of HCN was found high in young and early growth stages, yet it decreased and crude fiber increased in mature and advanced growth stages demonstrated impact on quality of forage sorghum when these were sown individually or together with forage legumes.Quality and quantity of forage sorghum intercropped with forage legumes, was significantly higher as there was better mixed green forage yield obtained when forage sorghum was grown in 30 cm apart rows and cluster bean was in between the rows [24].Another study was done on response of sorghum varieties JS-263 (old cultivar), JS-2002 and Chakwal Sorghum (recent cultivars) on different levels of soil moisture using physiological and morphological traits.At high soil moisture JS-2002 showed higher potential than Chakwal Sorghum while JS-263-old genotype showed insufficiency to face drought. .Six sorghum parent varieties (V-1, SV-6, CVS-13, SPV-462, RARI-S-10, TSS-9) and nine crosses were evaluated for fodder yield by heterosis and combining ability, revealing significant difference for all the traits under assessment.This study [27] indicated that a significant improvement in fodder yield of crosses is possible due to better and robust performance of these genotypes than parent heterosis along with good general combiner parents (V1, CVS-13).One of such sorghum line TSS-9 was selected for number of tillers per plant, while V-1 presented highest GCA for plant height, and CVS-13 was noted for stem thickness, fresh weight per plant, and dry weight per plant [27].In a study eight sorghum varieties (JS-2002, JS-263, MR-Sorghum-2011, Hegari, Pak-China-1, Sandal Bar, F-7017 and F-114) compared for yield and other quality related attributes, showed significant difference in dry matter yield, morphological traits, forage yield, and quality parameters (Table 1 1).The result suggested that frequent irrigation showed negligible impact on biomass and ethanol yield, whereas, water stress with 50% depletion reduced biomass per unit of water with no increase in sugar concentration or accumulation at harvest of sweet sorghum [51].
Wortmann and workfellows [52] evaluated sweet sorghum, corn, and grain sorghum for energy use efficacy, along with greenhouse gases (GHG) emissions and ethanol yield at seven different locations in Nebraska, USA.Grain crops showed 21% to 33% more calculated ethanol yield and net energy yield than sweet sorghum, although mean net energy yield of an earlier-maturing sweet sorghum cultivar was equivalent to grain crops.The total energy utilized to convert grain crops to ethanol was 23% lower than that utilized on sweet sorghum.However, sweet sorghum and grain crops reduced GHG emissions by 69% than gasoline.The byproducts of grain crops during ethanol production were utilized efficiently, while sweet sorghum bagasse were returned to field as soil fertilizers.2).Mature miRNA can be accumulated in specific tissues and organs of sugarcane.
The findings determined 46 potential targets for 19 miRNA of sugarcane several targets of conversed miRNA were involved in plant development as transcription factors.The findings classified 19 miRNA precursors in sugarcane and one miRNA precursor in sorghum into 14 families.Comparative analysis of sugarcane with sorghum showed common homologous miRNA and their targets in genome of these two species.Hence, such conservation may help to clarify specific aspects of miRNA regulation and evolution in the polypoid sugarcane [89].Foxtail millet (Setaria italica) belongs to poaccace family and is widely used as food, fodder and as model crop for biofuel grasses.Zhang et al.
[90] drafted genome onto nine chromosomes and annotated 38,801 genes.The findings demonstrated that reshuffling events of foxtail, sorghum and rice contributed in divergence of the crops.Two reshuffling events of key chromosomes were identified via collinearity between 2 and 9 chromosome of foxtail millet with 3, 7, 9, and 10 chromosomes of rice that occurred after divergence of foxtail and rice.Single reshuffling event occurred between foxtail millet chromosome 3 with rice chromosome 5 and 12, after divergence of sorghum and millet [90].Recent study showed genetic improvement of switchgrass biomass to develop emerging bioenergy crops.miR156 precursor was overexpressed in switchgrass and its effect on squamosa promoter binding protein like (SPL) genes were determined via microarray and RT-PCR.The findings characterized biomass yield, forage digestibility, saccharification efficiency and morphological alterations.miR156 overexpression suppresses SPL gene and is associated with apical dominance and transition in flowering time; while, low expression of miR156 was sufficient to increase biomass with normal flowering time, and disruption of apical dominance.Moderate expression of miR156 improved biomass, yet inhibits flowering.Thus, low and moderate expressions contribute in 58%-101% high biomass yield, though high miRNA expression result causes stunted growth (Table 2).Consequently, the degree of morphological alterations depends on level miR156 expression.High expression enhances solubilized sugar yield, forage digestibility and biomass yield by increasing tiller number [91].Switchgrass dedicated biofuel crop is broadly cultured for its high adaptability for marginal lands and high biomass yield.However, limited knowledge is available on basic mechanism of its gene expression under stressful conditions.Study conducted by Sun et al.
[92] demonstrated expression of regulatory miRNA on physiological parameters under salt and drought stress.The finding indicated that 1% salt stress adversely effected germination rate and growth, whereas, drought stress showed slight impact on germination.The miRNA expression of switchgrass under salt and drought stress was dose-dependent, and upregulated the gene expression by 0.9 folds and downregulated by 0.7 folds.Though, miRNA under both stresses showed similar range of expression yet, miRNA were more sensitive in drought stress, as miR156 and miR162 displayed significant expression, suggesting role of miRNA to cope up with drought stress (Table 2).Therefore, transgenic lines can improve bioenergy crops for biofuel production [92].Drought spells are widely known to reduce the yield of majority crops.Certain genes are expressed to plant cope up with drought stress and manage water, yet this mechanism remains unexplored in majority of crops.Ferreira et al.
[93] conducted a study to explore expression of miRNA in two cultivars of sugarcane under drought stress.Sugarcane cultivar RB855536 known for low drought tolerance and RB867515 cultivar with high tolerance were grown for 3 months and subjected to drought stress for 2-8 days.The results revealed identification of 18 miRNA families via deep sequencing, out of which 7 miRNA were differentially expressed in drought stress.Furthermore, differential expression of miRNAs depends on duration of stress, such as 6 miRNAs were differentially expressed after 2 days and 5 miRNA were expressed at 4 days of stress studied foxtail millet and identified gene families responsible for monolignol biosynthesis (PAL, C4H, 4CL, HCT, C3H, CCoAOMT, F5H, COMT, CCR, CAD), callose (Gsl) and cellulose (CesA/Csl).Comparative analysis of lignocellulose biosynthesis genes of foxtail millet with genome of C4 crops showed high resemblance with switchgrass followed by sorghum and maize.Whereas, expression profiling revealed that lignocellulosic gene was differentially expressed under abiotic stresses and hormonal treatments.The results suggested that monolignol biosynthesis proteins were highly diverse, while Gsl and CesA/Csl proteins were homologous to Oryza sativa and Arabidopsis thaliana [97].Furthermore, lignocellulosic crops faces economic barrier during biofuel production due to cell wall recalcitrance.Many researchers are actively working to explore genes that can offer solution to fix this problem via genome wide study.miRNAs are known to be involved in all biological, developmental and metabolic processes due to broad functions of their targets.Alteration in miRNA expression can lead to pleiotropic effects.Such as miRNA regulates physiological and biological traits such as low expression of miR156 increases biomass and reduces recalcitrance, while high expression of miRNA in bioenergy switchgrass and poplar reduces lignin content, increase biomass and flowering time with improved responses toward harsh environment [98].

42]. Table 1. Sorghum based research in Pakistan S. No. Sorghum variety Purpose Result Refs.
[15] 12. JS-2002, JS-263, Hegari-Sorghum, PSV-4, Sandalbar, Noor, FJ-115 Salinity tolerance JS-2002 and Sandalbar (tolerant), Hegari-sorghum 33.Ten genotypes of Sorghum: FS-08 FSD-11 NOOR F-114 F-113 NARC-11 AARI-10 AARI-08 FA-08 Drought tolerance NARC-11 and Sorgh-11 were drought tolerant F-114 was drought sensitive [36] Research on biofuel production from Sorghum worldwide Sweet sorghum is an emerging, potential candidate to serve for biofuel production due to its vast adaptability, easy cultivation, and high yield potential.Intensive exploitation of its diverse germplasm in various breeding programs has led to improved syrup, grain and forage yield.Monk et al. [43] performed study on increasing yield of sorghum hybrid in USA.The study demonstrated that both stalk and grain of this crops can contribute in energy sector such as 60 Mg ha−1 of fresh biomass can produce 5000 liters ha−1 of ethanol [43].

Table 3 . Role of miRNA in Sorghum
Use of sorghum biomass for energy purposes is of particular importance today because it allows reduction of greenhouse gases.Bioethanol is by far the most used biofuel for transport around the world.Although identification of potential miRNAs feedstocks have improvised and enhanced bioethanol production, one area of research possible in the future could explore the role of miRNAs in the factors of digestion, including the possible manipulation of miRNAs in Sorghum bicolor L.) by exogenous supply of salicylic acid.Pak.J. Bot 42(5): 3047-3054.10.Nawaz K, Talat A, Hussain K & Majeed A (2010).Induction of salt tolerance in two cultivars of sorghum (Sorghum bicolor L.) by exogenous application of proline at seedling stage.World Applied Sciences Journal 10(1): 93-99.11.Akram Z, Khan MM, Shabbir G & Nasir F (2011).Assessment of genetic variability in sorghum genotypes for drought tolerance based on RAPD analysis.J. Saleem A, Javed HI, Saleem R, Ansar M & Zia MA (2011).Effect of split application of potash fertilizer on maize and sorghum in Pakistan.Pakistan J. Agric.Res 24(1-4).16.Kausar A, Ashraf MY, Ali I, Niaz M & Abbass QAISER (2012).Evaluation of sorghum varieties/lines for salt tolerance using physiological indices as screening tool.Pakistan Journal of Botany 44(1): 47-52.17.Waqas HM, Firdous A, Ilyas M, Ashraf M, Zaka S, Zafar M & Shaheen Z (2012).Effect of NiCl on the Morphology of Sorghum bicolor.18. Shehzad M, Ayub M, Ahmad AUH & Yaseen M (2012).Influence of priming techniques on emergence and seedling growth of forage sorghum (Sorghum bicolor L.).J Ani Plant Sci 22: 154-158.19.Bibi A, Sadaqat HA, Tahir MHN & Akram HM (2012).Screening of sorghum (Sorghum bicolor var Moench) for drought tolerance at seedling stage in polyethylene glycol.J. Anim.Plant miR166a,b,c,d,e,f, miR167a,b,c,d,e,f,g, miR168, miR169a,b,c,d,e,f,g,h,i, miR171a,b,c,d,, miR172a,b,c,d,e,